Antiproliferative peptides and antibodies for their detection

ABSTRACT

The present invention relates to developmental peptides and peptidomimetics thereof, which may be used therapeutically to inhibit abnormal cell proliferation of damaged cells, including cancer cells or virally infected cells. In one embodiment, a seven to eleven amino acid developmental peptide and methods of using the same is provided.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.11/181,331 filed Jul. 14, 2005, which claims priority to U.S.Provisional Application No. 60/587,919 filed Jul. 14, 2004, the contentsof which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to peptides and peptidomimetics, which maybe used therapeutically to inhibit abnormal cell proliferation. It isbased at least in part, on the discovery of a class of developmentalpeptides or proteins (DPs) isolated from embryonic tissues which havebeen found to exhibit antiproliferative effects on a variety of damagedcells ranging from pre-cancerous to resistant cancer cells and virallyinfected cells. Developmental peptides and molecules that mimic thestructure and function of these peptides are capable of inhibitingabnormal cell proliferation.

In addition, the present invention relates to specific antibodiesgenerated against developmental peptides and their functionalequivalents, their use to determine the peptide expression in variousnormal and pathologic tissues and biological fluids, and their use asbiomarkers specifically but not limited to cancer. The present inventionfurther investigates the use of these antibodies for isolating andcharacterizing additional developmental peptides exhibitingantiproliferative action that may share homology to developmentalpeptides described herein.

The present invention relates to developmental peptides andpeptidomimetics which exert antiproliferative effects. The developmentalpeptides antiproliferative effects may be attributed to their ability tocontrol the delicate balance between proliferative and antiproliferativefactors in developing tissues within the embryo, and actually continuingsuch function throughout adulthood. Developmental peptides may furtherhave a similar antiproliferative homeostatic role in the adult,preventing abnormal proliferation, such as it occurs in cancer.

The discovery is based on the theory that pregnancy can be viewed as areversible form of cancer. In that context, embryonal cells are rapidlyproliferating, migrating, and invading the maternal body. In contrast tocancer cells, the embryonal cells undergo differentiation, give rise toorgan development and to a major shift from structure to functionleading to the birth of an individual. In the case of cancer, alteredcell proliferation leads in general to disrupted function andconsequently to the demise of the individual, unless the cancer isaggressively controlled and eradicated. An additional aspect ofpregnancy is that of maternal immune tolerance, which is a reversiblephenomenon and conditional in general on the health of the conceptus. Incontrast, frequently, the presence of cancer is associated with immunesuppression and altered immune response.

As described in U.S. Pat. No. 5,648,340 entitled “Gestational agents forcontrolling cell proliferation” filed Jan. 17, 1995 and PCT ApplicationNo. PCT/US91/08046 filed Oct. 31, 1991, entitled “Proteins Purified fromMammalian Gestational Tissue which Controls Cell Proliferation”incorporated herein by reference, developmental peptide agents have beenbroadly identified to operate by controlling the development of theembryo such that proliferation, placental invasivity and differentiationmay occur without substantially injuring the maternal host. U.S. Pat.No. 5,648,340 discloses the purification of protein extracts having amolecular weight less than 10,000 (and particularly less than 8,000daltons) which have antiproliferative activity and other agents of lessthan 3,000 daltons, which oppositely exhibit proliferative activity. Theprotein preparations described therein are described as high molecularweight extracts effective against a wide variety of viruses, as well asisolation and sequencing of a low molecular weight seven amino acid setof peptides which exert antiproliferative effects. In U.S. applicationSer. No. 10/117,728 filed Apr. 4, 2002 entitled “Gestational agentswhich modulate cell proliferation,” the profound and multi-targetedeffects of high molecular weight developmental peptides on cancer cellsproliferation is described. Developmental peptides inhibited tumorpromoters and promoted tumor inhibitors acting on both cell cycle andcell cycle independent intracellular proteins, with minimal effectexerted on normal white blood cells. Overall effect of developmentalpeptides appeared to be exerted at the G0-G1 transition phase. On MCF7cells (estrogen receptor positive breast cancer cells), P53phosphorylation increased while pRb decreased, mdm2 separated from p53,and later p21 was induced. Cyclin D1 and E were blocked, MAPkinasetemporarily dephosphorylated, Bcl2 was blocked while BAD increased.

The present invention relates to the further isolation andcharacterization of several developmental peptides as well as sequencingof such peptides, for example, from mammalian adult liver, anddocumenting their presence in a native form also within the porcineembryonal liver. In addition, generation of synthetic developmentalpeptides and peptidomimetics and testing of their activity againstcancer cells and virally infected cells and the mechanism ofdevelopmental peptide action is further described. Further, the presentinvention relates to the use of developmental peptides and KLH ascarrier for the generation of polyclonal antibody in chicken (IgY) anddetermining the expression of the proteins and peptides that contain aconserved seven to eleven amino acid sequence in a variety of embryonaland adult tissues.

The identification of developmental peptides in both fetal and adulttissues and their highly preferential expression in normal epitheliumpoint to their important biological role in rapidly replicating cellswhere negative regulators are likely to have a significant role inassuring proper control of proliferation. Also their expression inhighly proliferating and invasive cells as seen in the first trimestertrophoblast further confirms its in vivo role in maintaining control ofproliferation. On the other hand, their increased expression in tumorsassociated with an altered developmental peptide profile support theview that altered expression as a cause or consequence of malignanttransformation and thereby, the use of developmental peptides asbiomarkers, or therapeutics is envisaged. Developmental peptides appearto act through specific receptors that are expressed by abnormal and notnormal cells. As such, as seen in the embryo, developmental peptidesappear to act locally on the receptor target aimed in eliminatingabnormal cells. As such, homeostasis in the body is maintained wherenormal cells secrete and abnormal cells respond to developmentalpeptides. One aspect of the present invention provides a method foridentifying the developmental peptide receptor and creating apharmacophore to examine developmental peptide/receptor interaction.

SUMMARY OF THE INVENTION

One aspect of the present invention provides for developmental peptides,peptidomimetics and methods of their use for inhibiting abnormal cellproliferation.

Thus, according to one aspect of the invention, a syntheticdevelopmental peptide is provided, which binds to a developmentalpeptide receptor and inhibits abnormal cellular proliferation. Thedevelopmental peptide is preferably a seven to eleven amino acidpeptide. In preferred embodiments, the developmental peptide comprisesthe sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.1). In otherembodiments, the developmental peptide comprises the sequence ofN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2),N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.3),N-Ile-Asp-Val-Leu-Gly-LIys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.4),N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.5),N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.6),N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.7), orN-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.8),Gly-Lys-Arg-Ile (SEQ ID No.9), or Lys-Gly-Thr. In further embodiments,the developmental peptide has the sequenceXaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m), and Xaa_(n), eachrepresent an amino acid and wherein m independently has a value from 0to 20, preferably less than 10, and wherein n independently has a valuefrom 0 to 20, preferably less than 10, or mimetics thereof. In furtherembodiments, the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m), and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof.

The present invention also provides for non-peptide or partial peptidemimetic of any of the aforementioned developmental peptides.

In another aspect, the present invention provides for a method ofinhibiting abnormal cellular proliferation comprising administering theaforementioned developmental peptides or peptidomimetics in an amountsufficient to inhibit abnormal proliferation. In another embodiment, thecell is disposed within a living organism, preferably a mammal, morepreferably a human.

In a further aspect of the invention, a compound that binds todevelopmental peptide receptors and inhibits abnormal cell proliferationis provided. The compound has the formula R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH,wherein R₁ is Gly or a mimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃is Arg or a mimetic of Arg, R₄ is Ile or a mimetic of Ile, R₅ is Lys ora mimetic of Lys, R₆ is Gly or a mimetic of Gly and R₇ is Thr or amimetic of Thr. In alternative embodiments, the compound may comprisethe formula X-R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH, wherein X may comprise two tofour amino acid residues or mimetics of said residues, R₁ is Gly or amimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃ is Arg or a mimetic ofArg, R₄ is Ile or a mimetic of Ile, R₅ is Lys or a mimetic of Lys, R₆ isGly or a mimetic of Gly and R₇ is Thr or a mimetic of Thr. For example,X may comprise the sequence Val-Leu, Ile-Glu-Val-Leu, Ile-Asp-Val-Leu,Ile-Arg-Val-Leu, Ile-Glu-Val-Thr, Ile-Asp-Val-Thr, Ile-Arg-Val-Thr, ormimetics thereof.

The present invention also provides for a methodology for isolatingdevelopmental peptides from adult and embryonal tissues that haveselective antiproliferative effects on damaged cells, including, forexample, cancerous cells and virally infected cells, as compared tonormal cells. This methodology in one of its non-limiting embodimentsallows for isolation and identification of developmental peptidescontaining a seven amino acid sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH(SEQ ID No.1) present in both embryonal and adult mammalian tissue. Morepreferably, the developmental peptide is of the sequenceN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) that is presentin both embryonal and adult mammalian liver. The developmental peptidemay be isolated using chromatographic techniques or other isolationtechniques known in the art.

In another embodiment, a method for identifying and analyzingdevelopmental peptides and developmental peptide-like sequences within aprotein database is provided. Preferably, the analysis is performed onVal-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr (SEQ ID No.2) versus the humanprotein database.

A further embodiment of the present invention relates to thecharacterization and sequencing of a peptide that has significantantiproliferative effects on mammalian cancer cells. Preferably thedevelopmental peptide has a sequence Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n),wherein Xaa_(m) and Xaa_(n) each represent an amino acid and wherein mindependently has a value from 0 to 20, preferably less than 10, andwherein n independently has a value from 0 to 20, preferably less than10, or mimetics thereof. In further embodiments, the developmentalpeptide has the sequence Xaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m)and Xaa_(n) each represent an amino acid and wherein m independently hasa value from 0 to 20, preferably less than 10, and wherein nindependently has a value from 0 to 20, preferably less than 10, ormimetics thereof.

For example, the present invention relates to demonstrating thedevelopmental peptide antiproliferative effect against various cancercells including but not limited to breast and androgen receptor negativeprostate cancer cells. In a further non-limiting embodiment, the presentinvention relates to demonstrating developmental peptideantiproliferative effect against various virally infected cells. Whilenot wanting to be limited by theory, the antiproliferative effect may beexerted by blocking protein synthesis and creating mitochondrialcollapse. The antiproliferative effect could also be exerted againstleukemia and cancerous cells of the lung, liver, kidney, ovary, uterus,colon and the like.

Further, methods of inhibiting cancer cell proliferation comprisingadministering an effective amount of developmental peptides orpeptidomimetic thereof to a subject in need of such treatment. As such,the proteins, peptides and peptidomimetics of the invention may beuseful for the treatment of cancer and other proliferative disorders. Ina further embodiment, a method of potentiating chemotherapeutic agentsby administering a developmental peptide or mimetic thereof is provided.Methods of inhibiting viral replication and proliferation comprisingadministering an effective amount of developmental peptides or apeptidomimetic thereof to a subject in need of such treatment. Inaddition, administration of developmental peptides before and duringpregnancy may help reduce/eliminate teratogenicity/toxicity of differentcompounds or exposures.

In a further embodiment of the present invention, a method of generatingsynthetic developmental peptides that exhibit the same or substantiallysimilar activity of the native peptide is provided. Preferably, thesynthetic developmental peptides are of 70% homology or greater to thenative peptide. In preferred embodiments, the developmental peptidedevelopmental peptide has the sequence Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n),wherein Xaa_(m) and Xaa_(n) each represent an amino acid and wherein mindependently has a value from 0 to 20, preferably less than 10, andwherein n independently has a value from 0 to 20, preferably less than10, or mimetics thereof. In further embodiments, the developmentalpeptide has the sequence Xaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m)and Xaa_(n) each represent an amino acid and wherein m independently hasa value from 0 to 20, preferably less than 10, and wherein nindependently has a value from 0 to 20, preferably less than 10, ormimetics thereof.

In another embodiment of the present invention, a method of identifyingspecific receptor sites present on various tissues and cells that bindto developmental peptides with the effects being transduced by bindingto the developmental peptide receptor sites is provided.

In a further embodiment, a method of identifying and cloning the genesthat are responsible for developmental peptide expression, includingN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) or relatedpeptides.

The present invention also provides for pharmaceutical compositionscomprising said developmental peptides and peptidomimetics thereofisolated by the described methodology. Administration of thedevelopmental peptides or mimetic may be carried out using oral,enteral, parental or topical administration, including, for example,intravenous, oral, transdermal or any other mode of administration withappropriate vehicle. The developmental peptides may be used alone or incombination with other agents like chemotherapy, or immune basedtherapy.

Another embodiment of this invention is the isolation and cloning ofdevelopmental peptide receptors or related proteins that transduce thepeptide or in general developmental peptide effects. This method alsoprovides for identifying the intracellular mechanisms including but notlimited to the transcription factors that lead to the changes noted inprotein synthesis and mitochondrial collapse. Also the method may allowfor the identification of the developmental peptide secretory productsthat are modified following exposure to developmental peptides. It alsoprovides the method for identifying the genes' expression that aremodified secondary to exposure to the peptide.

In an additional embodiment, the present invention relates to method ofgenerating an antibody against developmental peptides and testing theantibody using immunohistochemistry in tissue arrays of humans andmouse. Assays could be used to monitor tumor presence, viral infectionand response to therapy in both tissues and isolated cells. The assaysprovide a method for determining embryonal health during pregnancy andmonitoring pregnancy well being. Specifically, the method providesdetermination of endogenous developmental peptide expression and itsrelation to cell proliferation, invasion and differentiation undernormal and pathological conditions. The method is applicable for alldevelopmental peptides and may be used to provide a diagnostic methodthat reflects the body homeostasis in all mammals.

In an additional embodiment, the present invention relates to a methodfor biomarker discovery by identifying developmental peptides inbiological fluids such as serum, urine, saliva, ascites. This methodentails generating affinity columns using anti-developmental peptideantibodies and examining collected samples by mass spectrometry, gelelectrophoresis or by chromatographic methods. Use of deuterium labeleddevelopmental peptides as internal standards may allow for evaluatingprecise recovery of each sample.

In another embodiment, the present invention relates to a method usingthe developmental peptide antibody for isolating and characterizingdevelopmental peptides that share a sequence homology with developmentalpeptide has the sequence Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), whereinXaa_(m) and Xaa_(n) each represent an amino acid and wherein mindependently has a value from 0 to 20, preferably less than 10, andwherein n independently has a value from 0 to 20, preferably less than10, or mimetics thereof; or the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof, more preferablyN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) by any methodincluding, but not limited to, affinity chromatography, Western blot, 2D gel electrophoresis, and mass spectrometry. In addition,identification of the pro-protein that encompasses the sequence willallow identification of the genes responsible for the encoding peptideand identify steps involved in the protein synthesis and processing thatappears to be altered in cancer. In one non-limiting embodiment, thecurrent method will allow for identification of protein to proteininteractions and identification of functions of several human endogenousretroviral particles (HERVs). As such, the current method structureprovides a method to determine the functional relationship betweencontrollers of proliferation and fundamental cellular structures thatare critical for cell survival such as gamma actin and the like. Alsothese methods allow for identification of the human gene and cloning ofthe gene that is responsible for developmental peptides and relatedproteins, as well those that interact with developmental peptides.

The present invention provides a screening method using mammalianembryos and labeled developmental peptides to determine whethercompounds are teratogenic, toxic, mutagenic, carcinogenic or infectious.As such it could provide a cost effective and sensitive method toeliminate potentially noxious compounds from the environment. As suchthe method could be used also for biodefense purposes to examinedevelopmental peptides binding to other cells such as in saliva todetermine whether exposure to a potentially toxic or infectious agenthas occurred

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one photograph or drawingexecuted in color. Copies of this patent with color drawing(s) orphotograph(s) will be provided by the Patent and Trademark Office uponrequest and payment of necessary fee.

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings.

FIG. 1 shows that cells isolated from renal cancer specimen werecompletely inhibited by addition of highly purified adult porcine liverderived developmental peptides <3000 kDa. In contrast, two type ofchemotherapy even in high doses had no effect. Developmental peptideseffect cause collapse of the cells and loss of cell to cell contact. A99% dose dependent inhibition of cells derived from a metastatic stageIV tubal epithelial carcinoma surgical sample was also demonstrated(data not shown).

FIG. 2 shows that cells isolated from renal cancer specimen werecompletely inhibited by addition of highly purified adult porcine liverderived developmental peptides <3000 kDa. In contrast, other types ofchemotherapy even in high doses had no effect. P1 and JC-1 staining bothshows >70% inhibition, while the most effective chemotherapy effect was20%.

FIG. 3 illustrates the anti-angiogenic effect of highly purifieddevelopmental peptides<3000 daltons isolated from adult porcine liverupon resistant androgen prostate cancer cells (PC3). developmentalpeptides blocked VEGF expression by cancer cells. This complements theblock of expression of NF kB in these cells (data not shown).

FIG. 4 shows microphotographs comparing the effects of highly purified<3000 daltons developmental peptides derived from adult porcine livercompared to controls. Images shows that in all cases, preneoplasticbreast, prostate, patients renal cancer exposure to developmentalpeptides causes practically collapse of the cell, without extrusion ofthe cellular content, a significant reason for chemotherapy inducedtoxicity.

FIG. 5 illustrates the sequence of the novel 970 daltons peptides (i.e.SEQ ID NOs. 3, 4, 5, 6, 7 and 8, respectfully) and the shared homologywith a portion of the retrovirus enzyme.

FIG. 6 shows that synthetic developmental peptides (9aa) are notteratogenic when added to rat embryo cultures for 48 hours, using theroler bottle system.

FIG. 7 shows that exposure of FITC-developmental peptides to rat embryoscultured in roler bottles for 48 hours was not associated with uptake bythe tissue of the peptides.

FIG. 8. In contrast, exposure to 5FU (a potentteratogen/chemotherapeutic agents) for 3 hours led to major uptake ofFITC-developmental peptides by the various tissues. This indicatesexpression of development peptides receptor only when the embryo isexposed to a teratogen/mutagen.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particulardevelopmental peptide versions or embodiments only, and is not intendedto limit the scope of the present invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The terms “mimetic,” “peptide mimetic” and “peptidomimetic” are usedinterchangeably herein, and generally refer to a peptide, partialpeptide or non-peptide molecule that mimics the tertiary bindingstructure or activity of a selected native peptide or protein functionaldomain (e.g., binding motif or active site). These peptide mimeticsinclude recombinantly or chemically modified peptides, as well asnon-peptide agents such as small molecule drug mimetics, as furtherdescribed below.

In one embodiment, the developmental peptides of the invention aremodified to produce peptide mimetics by replacement of one or morenaturally occurring side chains of the 20 genetically encoded aminoacids (or D amino acids) with other side chains, for instance withgroups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7 memberedalkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy,hydroxy, carboxy and the lower ester derivatives thereof, and with 4-,5-, 6-, to 7 membered heterocyclics. For example, proline analogs can bemade in which the ring size of the proline residue is changed from 5members to 4, 6, or 7 members. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or nonaromatic.Heterocyclic groups can contain one or more nitrogen, oxygen, and/orsulphur heteroatoms. Examples of such groups include the furazanyl,furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl,isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino),and triazolyl. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.Peptidomimetics may also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24, 243-252, 1989). Certain peptidomimetic compounds are basedupon the amino acid sequence of the peptides of the invention. Often,peptidomimetic compounds are synthetic compounds having athree-dimensional structure (i.e. a “peptide motif) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the peptidomimetic compound with the desired biologicalactivity, i.e., binding to IAP, wherein the binding activity of themimetic compound is not substantially reduced, and is often the same asor greater than the activity of the native peptide on which the mimeticis modeled. Peptidomimetic compounds can have additional characteristicsthat enhance their therapeutic application, such as increased cellpermeability, greater affinity and/or avidity and prolonged biologicalhalf-life.

Peptidomimetic design strategies are readily available in the art (see,e.g., Ripka & Rich, Curr. Op. Chem. Biol. 2, 441-452, 1998; Hruby etal., Curr. Op. Chem. Biol. 1, 114-119, 1997; Hruby & Balse, Curr. Med.Chem. 9, 945-970, 2000). One class of peptidomimetics a backbone that ispartially or completely non-peptide, but mimics the peptide backboneatom for atom and comprises side groups that likewise mimic thefunctionality of the side groups of the native amino acid residues.Several types of chemical bonds, e.g., ester, thioester, thioamide,retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, areknown in the art to be generally useful substitutes for peptide bonds inthe construction of protease-resistant peptidomimetics. Another class ofpeptidomimetics comprises a small non-peptide molecule that binds toanother peptide or protein, but which is not necessarily a structuralmimetic of the native peptide. Yet another class of peptidomimetics hasarisen from combinatorial chemistry and the generation of massivechemical libraries. These generally comprise novel templates which,though structurally unrelated to the native peptide, possess necessaryfunctional groups positioned on a nonpeptide scaffold to serve as“topographical” mimetics of the original peptide (Ripka & Rich, 1998,supra).

Thus, according to one aspect of the invention, a syntheticdevelopmental peptide is provided, which binds to a developmentalpeptide receptor and inhibits abnormal cellular proliferation. Thedevelopmental peptide is preferably a seven to eleven amino acidpeptide. In preferred embodiments, the developmental peptide comprisesthe sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.1). Abnormalcellular proliferation may include, for example, cancer or viralinfection.

Inhibition of abnormal cellular proliferation by developmental peptidesinvolves control of proliferation by elimination of damaged cellswithout interfering with normal cell proliferation and without beingtoxic. To eliminate damaged cells, developmental peptides may useseveral pathways and processes which involve a cascade to include knownpathways, simultaneously or sequentially, until the damaged cell isunable to replicate or transmit its damage, culminating in the damagedcell's death. Damaged cells include cancer cells or those cells infectedwith a virus.

In other embodiments, the developmental peptide comprises the sequenceof N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2),N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.3),N-Ile-Asp-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.4),N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.5),N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.6),N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.7), orN-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.8). Infurther embodiments, the developmental peptide contains the sequenceXaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m) and Xaa_(n), eachrepresent an amino acid and wherein m independently has a value from 0to 20, preferably less than 10, and wherein n independently has a valuefrom 0 to 20, preferably less than 10, or mimetics thereof. In furtherembodiments, the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof.

The present invention also provides for non-peptide or partial peptidemimetic of any of the aforementioned developmental peptides.

In another embodiment of the present invention, identification andsequence analysis of developmental peptides or developmentalpeptide-like molecules is performed on a known protein database.Preferably, a blast search is performedVal-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr (SEQ ID No.2) versus the humanprotein database.

A further embodiment of the present invention relates to thecharacterization and sequencing of a peptide that has significantantiproliferative effects on damaged mammalian cells, for example cancercells and virally infected cells. Preferably the developmental peptidecontains the sequence Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m)and Xaa_(n) each represent an amino acid and wherein m independently hasa value from 0 to 20, preferably less than 10, and wherein nindependently has a value from 0 to 20, preferably less than 10, ormimetics thereof. In further embodiments, the developmental peptide hasthe sequence Xaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n)each represent an amino acid and wherein m independently has a valuefrom 0 to 20, preferably less than 10, and wherein n independently has avalue from 0 to 20, preferably less than 10, or mimetics thereof. Forexample, the present invention relates to demonstrating thedevelopmental peptide antiproliferative effect against various cancercells including but not limited to breast and androgen receptor negativeprostate cancer cells. The antiproliferative effect could also beexerted against leukemia and cancerous cells of the lung, liver, kidney,ovary, uterus, colon and the like. The present invention also relates todemonstrating the developmental peptide antiproliferative effect againstvirally infected cells, including, but not limited to cells infectedwith retroviruses such as HIV. While not wanting to be limited bytheory, the antiproliferative effect may be exerted by blocking proteinsynthesis, blocking survival factors, promoting death signals andcreating mitochondrial collapse. Further the effect may be achieved byblocking oncogenic pathways, including but not limited to ras-raf, srckinase, and IP3 kinase, activating phosphatases, and blockingangiogenesis and blocking NFkB activity.

In a further aspect of the invention, a compound that binds todevelopmental peptide receptors and inhibits abnormal cell proliferationis provided. The compound has the formula R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH,wherein R₁ is Gly or a mimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃is Arg or a mimetic of Arg, R₄ is Ile or a mimetic of Ile, R₅ is Lys ora mimetic of Lys, R₆ is Gly or a mimetic of Gly and R₇ is Thr or amimetic of Thr. In alternative embodiments, the compound may comprisethe formula X-R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH, wherein X may comprise two tofour amino acid residues or mimetics of said residues, R₁ is Gly or amimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃ is Arg or a mimetic ofArg, R₄ is Ile or a mimetic of Ile, R₅ is Lys or a mimetic of Lys, R₆ isGly or a mimetic of Gly and R₇ is Thr or a mimetic of Thr. For example,X may comprise the sequence Val-Leu, Ile-Glu-Val-Leu, Ile-Asp-Val-Leu,Ile-Arg-Val-Leu, Ile-Glu-Val-Thr, Ile-Asp-Val-Thr, Ile-Arg-Val-Thr, ormimetics thereof.

The present invention also provides for a methodology for isolatingdevelopmental peptides from adult and embryonal tissues that haveselective antiproliferative effects on cancerous tissue and cancerouscells or virally infected cells as compared to normal cells. Thismethodology in one of its non-limiting embodiments allows for isolationand identification of developmental peptides containing a four aminoacid sequence of Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m) andXaa_(n) each represent an amino acid and wherein m independently has avalue from 0 to 20, preferably less than 10, and wherein n independentlyhas a value from 0 to 20, preferably less than 10, or mimetics thereof.In further embodiments, the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof, or a seven aminoacid sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.1) present inboth embryonal and adult mammalian tissue. More preferably, thedevelopmental peptide is of the sequenceN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) that is presentin both embryonal and adult mammalian liver. The developmental peptidemay be isolated using chromatographic techniques or other isolationtechniques known in the art.

The present invention also provides for pharmaceutical compositionscomprising said developmental peptides and peptidomimetics thereofisolated by the described methodology. Preferably the developmentalpeptide comprises the sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ IDNo.1), N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2),N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.3),N-Ile-Asp-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.4),N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.5),N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.6),N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.7), orN-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.8). Infurther embodiments, the developmental peptides contain the sequenceXaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m) and Xaa_(n) eachrepresent an amino acid and wherein m independently has a value from 0to 20, preferably less than 10, and wherein n independently has a valuefrom 0 to 20, preferably less than 10, or mimetics thereof. In furtherembodiments, the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof.

The developmental peptides and mimetics may be administered in aneffective amount to a subject in need of such treatment. As such, theproteins and peptides described herein may be useful for the treatmentof cancer and other proliferative disorders, including viral infections.Administration of the developmental peptides or peptidomimetic, in theform of a therapeutic agent, may be carried out using oral, enteral,parenteral or topical administration, including, for example,intravenous, oral, transdermal or any other mode of administration withappropriate vehicle. The developmental peptide or mimetic thereof mayused alone or in combination with other agents.

Pharmaceutical compositions can be used in the preparation of individualdosage forms. Consequently, pharmaceutical compositions and dosage formsof the invention comprise the active ingredients disclosed herein (i.e.,developmental peptides, or mimetics thereof). Pharmaceuticalcompositions and dosage forms of the invention can further comprise oneor more excipients.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms encompassedby this invention will vary from one another will be readily apparent tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise anamount of from about 1 mg to about 2000 mg, more preferably from about50 mg to about 1000 mg, even more preferably from about 100 mg to about750 mg, and more preferably from about 200 mg to about 500 mg.

In another aspect, the present invention provides for a method ofinhibiting abnormal cellular proliferation comprising administering theaforementioned developmental peptides or peptidomimetics in an amountsufficient to inhibit abnormal proliferation. In another embodiment, thecell is disposed within a living organism, preferably a mammal, morepreferably a human.

Further, methods of inhibiting cancer cell proliferation comprisingadministering an effective amount of developmental peptides orpeptidomimetics of said developmental peptides to a subject in need ofsuch treatment. Additionally, methods of inhibiting viral replicationand proliferation comprising administering an effective amount ofdevelopmental peptides or peptidomimetics of said developmental peptidesto a subject in need of such treatment. As such, the proteins, peptidesand peptidomimetics of the invention may be useful for the treatment ofcancer, viral infections and other proliferative disorders.

Developmental peptides could be used alone or in combination withchemotherapeutic agents or immunotherapies. Developmental peptides maybe administered orally, parenterally, enterally, nasally, transmucosallyor by inhalation and the like. Developmental peptides may exert theirantiproliferative effects against cancer, including, but not limited tocancers of the prostate, kidney, fallopian tube, or breast. Such cancercan be early stage or advanced form, including metastatic cancer. Thedevelopmental peptides can be given also for the treatment preneoplasticlesions or as prevention for the development of malignancy. Thereforeone of the embodiments of the invention is the use of developmentalpeptides to combine with chemotherapy to lower the toxicity of achemotherapeutic agent by allowing for a reduction in the doses of thecurrently used chemotherapeutic agents.

The developmental peptides, or mimetics thereof, are preferablyadministered in effective amounts. An effective amount is that amount ofa preparation that alone, or together with further doses, produces thedesired response. This may involve only slowing the progression of thedisease temporarily, although preferably, it involves halting theprogression of the disease permanently or delaying the onset of orpreventing the disease or condition from occurring. This can bemonitored by routine methods. Generally, doses of active compounds wouldbe from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expectedthat doses ranging from 50-500 mg/kg will be suitable, preferablyintravenously, intramuscularly, or intradermally, and in one or severaladministrations per day. The administration of the developmentalpeptides, or mimetics thereof, can occur simultaneous with, subsequentto, or prior to chemotherapy or radiation so long as thechemotherapeutic agent or radiation sensitizes the system to thedevelopmental peptides, or mimetics thereof.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect for each therapeuticagent and each administrative protocol, and administration to specificpatients will be adjusted to within effective and safe ranges dependingon the patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient, the developmentalpeptides, or mimetics thereof, potencies, the duration of the treatmentand the severity of the disease being treated. For example, a dosageregimen of the developmental peptides, or mimetics thereof, can be oraladministration of from 1 mg to 2000 mg/day, preferably 1 to 1000 mg/day,more preferably 50 to 600 mg/day, in two to four (preferably two)divided doses, to reduce tumor growth. Intermittent therapy (e.g., oneweek out of three weeks or three out of four weeks) may also be used.

In the event that a response in a subject is insufficient at the initialdoses applied, higher doses (or effectively higher doses by a different,more localized delivery route) may be employed to the extent that thepatient tolerance permits. Multiple doses per day are contemplated toachieve appropriate systemic levels of compounds. Generally, a maximumdose is used, that is, the highest safe dose according to sound medicaljudgment. Those of ordinary skill in the art will understand, however,that a patient may insist upon a lower dose or tolerable dose formedical reasons, psychological reasons or for virtually any otherreason.

In another non-limiting embodiment, the present invention relates todemonstrating the developmental peptide antiproliferative effect againstvarious cancer cells, including, but not limited to, breast, andandrogen receptor negative prostate cancer cells. The antiproliferativeeffect is dose dependent and the effective concentration shows nonotable effect on normal cells or embryos. While not wanting to belimited by theory, the antiproliferative effect may be exerted byblocking protein synthesis and creating mitochondrial collapse. Theantiproliferative effect could also be exerted against leukemia cells,or abnormal cells of the lung, liver, kidney, ovary, uterus, colon andthe like.

In a further embodiment, a method of potentiating chemotherapeuticagents by administering a developmental peptide or peptidomimeticthereof as described herein. For example, in the method, achemotherapeutic agent that alone was not effective or fully effective,will become effective when developmental peptides are added to thetherapy resulting in an antiproliferative effect. Such a method isuseful when the agents alone are not effective, or when treatment agentsare effective but are highly toxic, enabling their dose to be loweredand therefore toxicity could be limited or eliminated altogether.

In a further embodiment of the present invention, a method of generatingsynthetic developmental peptides that exhibit the same or substantiallysimilar activity of the native peptide is provided. Preferably, thesynthetic developmental peptides are of 70% homology or greater to thenative peptide. In preferred embodiments, the developmental peptide hasthe following sequence Xaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m)and Xaa_(n) each represent an amino acid and wherein m independently hasa value from 0 to 20, preferably less than 10, and wherein nindependently has a value from 0 to 20, preferably less than 10, ormimetics thereof. In further embodiments, the developmental peptide hasthe sequence Xaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n)each represent an amino acid and wherein m independently has a valuefrom 0 to 20, preferably less than 10, and wherein n independently has avalue from 0 to 20, preferably less than 10, or mimetics thereof.

In a further embodiment, a method of identifying and cloning the genesthat are responsible for developmental peptide expression. In apreferred embodiment, the gene identified and cloned is responsible forthe peptide of SEQ ID NO: 2.

In another embodiment of the present invention, a method of identifyingspecific receptor sites present on various tissues and cells that bindto developmental peptides and are transduced by binding to thedevelopmental peptides receptor sites is provided.

Another embodiment of this invention is the isolation and cloning ofdevelopmental peptide receptors or related proteins that transduce thepeptide or in general developmental peptide effects. This method alsoprovides for identifying the intracellular mechanisms including but notlimited to the transcription factors that lead to the changes noted inprotein synthesis and mitochondrial collapse. Also the method may allowfor the identification of the developmental peptide secretory productsthat are modified following exposure to developmental peptides, such as,but not limited to, cytokines and growth factors. It also provides themethod for identifying the genes' expression that are modified secondaryto exposure to the peptide. This embodiment also includes the use oflabeled developmental peptides for diagnostic purposes both in vitro andwhen administered in vivo.

The present invention provides a method for isolating and characterizinga novel developmental peptide antiproliferative peptide of SEQ ID NO: 2in one non limiting embodiment. Accordingly, the present inventionprovides for therapeutic compositions comprising the developmentalpeptide or a protein that contains the peptide prepared by chemicalsynthesis and for selectively inhibiting the proliferation of cancercells. In particular, in non-limiting embodiments, the present inventionprovides for antiproliferative compositions where the protein may haveanti-proliferative effects, for example but not limited to, in an assayof MCF-7 breast cancer cells and PC3 prostate cancer cells, whereproliferation is inhibited by at least 30% and preferably by at least75%, or by adding developmental peptides to a chemotherapeutic agent toenhance the efficacy of the anti-cancer treatment regimen by renderingthe chemotherapeutic agent effective against cancer in models andpatients where the chemotherapeutic agent failed to do so alone. Thecomposition may further comprise a suitable pharmaceutical carrier andoptionally one or more additional bioactive agent.

The agents of the invention could be used as antiproliferative agents toprotect from a pre-malignant to malignant transformation or decreaseproliferation of malignant cells. The antiproliferative effects may beproduced in vitro or in vivo. In particular, but not limiting,developmental peptides may be used to prevent or treat cancers involvingbreast, prostate, kidney, bone, liver, melanoma, colon, skin, testicle,and ovary among others. The compositions of the invention may thus beused to prevent or to inhibit the growth or spread of malignant cells ina subject in need of such treatment.

In one preferred current embodiment, the peptide isN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 2) or above 70%sequence homology. This sequence shares partial homology with the polprotein (a portion of the endogenous reverse transcriptase) of simiantype retrovirus. Such peptides could be modified by conjugation toanother compound where the compound is selected from the groupincluding, but not limited to, other proteins (e.g. immunoglobulinmolecules or fragments of), lipids and carbohydrate residues,pharmaceutical agents, polyethylene glycol, etc., or may incorporatedinto a larger peptide or protein (e.g., a fusion protein).

The present invention provides for isolated nucleic acids encoding thepeptides of the invention. Such peptides may be comprised in a suitablevector for cloning and or expression.

The present invention also provides for developmental peptides as setforth by producing combinatorial mixtures of the possible peptides. Thepeptides may be prepared from natural sources, chemically synthesized orproduced recombinant DNA methods. The present invention also providesfor the introduction, into a subject of a nucleic acid encoding one ormore of the foregoing peptides are expressed. The subject may be amicroorganism, such as bacterium, yeast, a eukaryotic cell, such asmammalian, insect, or plant cell, may be multicellular organism such asa mammal or bird.

The antiproliferative developmental peptides of the invention may beused in methods of inhibiting cell proliferation and particularlyinhibiting malignant cell proliferation. They may be administered in aneffective dose and in suitable pharmaceutical carrier to a subject inneed of such treatment. Administration methods include but are notlimited to topical, intravenous, intraperitoneal, intrapulmonary,intrathecal, subcutaneous as well as a local injection into a tissue ortumor. Proliferative conditions that may benefit from the administrationof the developmental peptides of the invention include, but are notlimited to, cancer, including but not limited to breast, prostate,kidney, colon, bladder, melanoma, leukemia, as well as pre-malignantlesions of different organs and hyperproliferative conditions such asrheumatoid arthritis and keloid formation.

Additional embodiment is using labeled developmental peptides as ascreening tool to examine effect of various mutagens/toxicagents/carcinogens and infectious agents on normal and developmentaltissues in a preclinical setting. This could be useful for theexamination of adverse environments, pollutants and agents for example(not to be limiting in any manner) in the development for clinical use,wherein positive results may lead to removal of the offending agents oravoidance of exposure to them, including biodefense. While in drugdiscovery it could be a rapid tool to modify compounds to limit theirtoxicity towards a more acceptable profile.

In an additional embodiment, the present invention relates to method ofgenerating an antibody against developmental peptides. The antibody maybe a monoclonal or polyclonal antibody, and further the antibody may beIgA, IgG, IgM, IgD, IgE, and more preferably IgY. In a furtherembodiment, the antibody may be tested using immunohistochemistry intissue arrays of humans and mouse. Assays could be used to monitor tumorpresence and response to therapy. The assays provide a method fordetermining embryonal health during pregnancy and monitoring pregnancywell being using various body fluids and tissues. The assays could beused to identify developmental peptide related biomarkers in various nonpregnant biological fluids as well by using ELISA, affinitychromatography or other non-limiting detection method. Identification ofaltered proteins/peptides related to developmental peptides due todisease would aid in the identification of other biomarkers. Usingdeuterium labeled developmental peptides as an internal standard, forexample in the valine and/or leucine amino acid, would help to monitorrecovery and sensitivity of the assay. The method is applicable for alldevelopmental peptides and may be used to provide a diagnostic method,as well biomarkers for disease, that reflects the body homeostasis inall mammals. Preferably, the antibody is generated against SEQ ID No.2.

In another embodiment, the present invention relates to a method usingthe developmental peptides antibody for isolating and characterizingdevelopmental peptides that share a sequence homology with developmentalpeptides, preferably of the amino acid sequenceXaa_(m)-Gly-Lys-Arg-Ile-Xaa_(n), wherein Xaa_(m) and Xaa_(n) eachrepresent an amino acid and wherein m independently has a value from 0to 20, preferably less than 10, and wherein n independently has a valuefrom 0 to 20, preferably less than 10, or mimetics thereof. In furtherembodiments, the developmental peptide has the sequenceXaa_(m)-Lys-Gly-Thr-Xaa_(n), wherein Xaa_(m) and Xaa_(n) each representan amino acid and wherein m independently has a value from 0 to 20,preferably less than 10, and wherein n independently has a value from 0to 20, preferably less than 10, or mimetics thereof, more preferredN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) by any methodincluding, but not limited to, affinity chromatography, Western blot, 2D gel electrophoresis, and mass spectrometry. In addition,identification of the pro-protein that encompasses the sequence willallow identification of the genes responsible for the peptide encodingand identify steps involved in the protein synthesis and processing thatappears to be altered in cancer. In one non-limiting embodiment, thecurrent method will allow for identification of protein to proteininteraction and identification of the function of several HERVs. Themethod also allows examination of the relationship between elementsinvolved in cell proliferation, differentiation and invasivity, such asuse of cell proliferation markers Ki67 and HSP 27 for example, in normaland pathologic samples. As such, the current method structure provides amethod to determine the functional relationship between controllers ofproliferation and fundamental cellular structures that are critical forcell survival such as gamma actin and the like. Also these methods allowfor identification of the human gene and cloning of the gene that isresponsible for developmental peptides and related proteins, as wellthose that interact with developmental peptides.

In another embodiment, the present invention relates to method ofgenerating a chicken antibody (IgY) against the developmental peptides,preferably SEQ ID No.2, and testing the antibody usingimmunohistochemistry in tissue arrays of humans and mouse. Thisembodiment is based, at least in part on the finding that the expressionof the peptide/protein that contains the peptide is present mostly inepithelial cells as well as pancreas where the highest expression wasnoted. The expression of developmental peptides using IgY was hundredsof fold higher in a 60 tumor panel epithelium than in normal tissues.For example, antibody production could be made in rabbits, goats andmice, poly and monoclonal, used as diagnostic methods in vitro as wellas in vivo for determining developmental peptide levels in biologicalfluids (such as blood, urine, saliva, embryo culture media and thelike), tissues and cells using ELISA, EIA or lateral flow assay as wellas the antibody can be injected to image tumors in the body as nonlimiting examples.

The assays could be used to monitor tumor presence, and response totherapy. The assays provide a method for determining embryonal healthduring pregnancy and monitoring pregnancy well being The method isapplicable for all developmental peptides and related proteins and maybe used to provide a diagnostic method that reflects the body'shomeostasis in all mammals.

In another embodiment, the present invention relates to a method usingthe IgY antibody or IgG monoclonal antibody for isolating andcharacterizing developmental peptides that share a sequence homologywith antiproliferative developmental peptides by any method including,but not limited to, affinity chromatography, Western blot, 2 D gelelectrophoresis, and mass spectrometry and the like. This is based onthe demonstration of differential expression between a normal and ahepatocarcinoma sample. Since the human placenta, analyzed using aWestern blot, has a similar protein expression profile as adult tissuethe methodology described using, in a non limiting manner, affinitychromatography allows for isolation of additional peptides that may havepotency, efficacy and selectivity of action that is even greater thanSEQ ID NO: 2. In addition, identification of the pro-protein thatencompasses the peptide will allow for the identification of genesencoding the peptide and also allow the identification of steps involvedin the protein synthesis and processing that appears to be altered incancer.

Using the IgY dependent affinity chromatography coupled with massspectrometry, sequencing suggested that the larger developmentalpeptides may be attached to cytoskeleton proteins and RNA polymerase andhuman endogenous retrovirus proteins within the cell. Therefore currentmethodology, in a non limiting embodiment, will allow for identificationof protein to protein interaction(s) and the identification of thefunction of several HERV. As such, the current method structure providesa method to determine the functional relationship between controllers ofproliferation and fundamental cellular structures that are critical forcell survival, such as gamma actin and the like. Also these methodsallow for identification of the human gene(s) and cloning of the gene(s)responsible for developmental peptides and related proteins, as wellthose that interact with developmental peptides.

These non limiting examples show that other developmental peptides mayalso be present both in fetal and adult tissues which sequences couldserve as diagnostics for documenting abnormal cell proliferation andcould be the basis for generation of specific antibodies IgY, IgG, andmonoclonal as non limiting examples. Further evidence is provided byfirst trimester placental explants where co-localization ofproliferation and invasivity markers (Ki67 and HSP27) was found withdevelopmental peptides. In this context developmental peptides couldlimit neoplastic like cytotrophoblast and extravillous trophoblastproliferation and invasion.

Developmental peptide antibodies could be used for biomarker discoveryand validation in biological fluids including serum, urine, saliva,ascites this by using developmental peptides detection through, forexample, developmental peptides ELISA, or affinity chromatography. Tothat end a developmental peptide (SEQ ID NO: 2) was generated usingdeuterium labeled Leu (mass+10) as one peptide and Val (mass+8) and Leu(mass 10) for a total 18 for the other. This is used to monitor peptiderecovery following affinity chromatography and mass spectrometry forquantitative assessment.

Also the analysis of tumor samples could be carried out by variouschromatographical methods using the antibody as well as 2D gelelectrophoresis, mass spectrometry, HPLC, etc., in a non limitingmanner. In addition presence of the antibodies could be used for thedevelopment of quantitative assays for measuring the concentration ofdevelopmental peptides and related proteins in biological samples andfluids, by using ELISA, lateral flow EIA, etc., as non limitingexamples.

The developmental peptides and peptidomimetics thereof in the presentinvention may be coupled to labels, including but not limited to FITC,biotin, rhodamine, radioactive labels, fluorescent nanocrystals, andother labels known to those skilled in the art. The labeleddevelopmental peptides could be used by introducing a labeleddevelopmental peptide molecule by injection into the body, labeling maybe with fluorescence, for such as but not limited to, rhodamin, biotin,etc. The injected molecule could be used to determine the localizationof tumors using radiographic and scanning methods to better diagnosedisease and direct therapy.

The labeled developmental peptides and peptidomimetics thereof that mayalso be used to identify specific receptor sites present on varioustissues and cells where the developmental peptide specifically binds aswell as the intracellular effects that are transduced by binding tothese receptors within the cell. The labeled developmental peptides canbe used also to document changes in expression of developmental peptidesin pathological conditions due to disease or due to exposure tocarcinogens/toxins and infectious agents.

Example 1 Isolation of Developmental Peptides and Preparation ofSynthetic Developmental Peptides

Isolation of the 9AA peptide from the adult mammalian liver. Fresh adultrabbit liver tissues were collected and homogenized on ice with H₂0. Thesamples were centrifuged at 15,000 g and the sample was filtered usingAmicon filters 3000-10000 Da, <3000-500, and <500. Fractions collectedwere tested for activity using the MCF-7 cells. The highestantiproliferative activity was noted at the 3-10 kDa region, theantiproliferative activity of developmental peptides was principallynoted in the early fractions with about 99% inhibition of breast cancercells (MCF-7). Separation by reverse phase HPLC yielded a 90% inhibitionof cancer cells.

In a similar manner adult frozen porcine liver was collected andhomogenized with H₂O at 4° C. The suspension was centrifuged at 15,000 gand the pellet discarded. The suspension was filtered and the fraction<3000 Da was collected and run using reversed phase HPLC C18 column witha 0-95% acetonitrile gradient. Separation of the active fractionrevealed several fractions, in particular at approximately 500-3000 Daregions, that displayed anti-proliferative activity. Antiproliferativeactivity was tested by collecting the fractions, drying and suspendingthe sample in media and adding to MCF7 cells for 48 hours. Incorporationof 3H thymidine into cellular DNA of MCF-7 breast cancer cells was usedto determine the anti-proliferative activity. The peak fraction that wasobserved to provide the most anti-proliferative activity was sequenced,yielding a 9 amino acid (AA) peptide of SEQ ID NO: 2.

Almost total inhibition of the cancer cell proliferation was observedwith the earlier fractions (fractions in the range of between the10^(th)-16^(th) fractions collected). The early bioactive fractions werefurther separated by a similar reversed phase HPLC, collected, drieddown and resuspended in culture media and tested on MCF7 cells. Thefraction that yielded the greatest inhibition was submitted forsequencing using the Edman degradation method. The sequence derived wasnine amino acid peptide of the sequenceN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 2).

In order to confirm that the peptide generated has a role in embryodevelopment an embryonal extract was prepared by solubilizing(homogenizing and/or forming a cell lysate) a mammalian porcine embryoliver. While in this example the porcine embryo liver was used, otherembryo samples may also be used. For example, the whole embryo may beused to prepare the embryo extract, including but not limited toembryo's derived from a human, pig, cow, horse, sheep or goat; or aportion of an embryo may be used such as the liver, placenta, brain,pancreas. The frozen embryo liver tissue was placed in liquid nitrogenand was grounded to fine powder and then transferred to a 50 ml Falcontube and 23 ml of C7 solubilizing solution was added. The solution wassonicated with a high power ultrasonic probe for 6×15 seconds (intensity60%) on ice. The sample was spun down at 21,000 g for 45 minutes in roomtemperature. The supernatant was removed and the pellet was frozen. To20 ml of the supernatant 500 ul of the reducing agent was added and 200ul of the alkylation agent. The solution was let stand for 90 minutes atroom temperature. The alkylation reagent was quenched by adding 200 ulQuenching agent. Acetonitrile (ACN) was added to precipitate proteins at1:5 final volume, such as 20 ml of sample and 80 ml CAN. Samples arespun at 3000 g for 5 minutes, room temperature. The pellet was dried atroom temperate for 10 minutes. The pellet was resuspended in Chapschamber solution. Pellet is allowed to slowly dissolve and proteinconcentration was determined by Lowry assay. 1 ml of solution was runovernight on MCE 16 hours at 1500 V Maximum. The cathode buffer fractionwas removed for CAN precipitation and MALDI analysis. A number ofpeptides 970-1230 daltons were identified. The 970 dalton developmentalpeptide was analyzed in detail, which was sequenced by using the Lasermass spec technique. The sequence identified wasN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 2) the same as thatof the adult liver.

Synthetic peptide preparation. The derived 970 dalton developmentalpeptide sequence was used to generate synthetic peptideN-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) using solid-phasepeptide synthesis (SPPS), carried out on an Applied Biosystems Model4331A Peptide Synthesizer employing Fmoc chemistry in which the α-aminonitrogen of each amino acid is blocked with Fmoc(9-fluorenylmethoxycarbonyl). Three equivalents of the C-terminal aminoacid are added to 1 equivalent of Wang resin (p-benzyloxybenzyl alcoholattached to a polystyrene resin) in the presence ofN,N-dicyclohexylcarbodiimide to form the anhydride. Following couplingof the first amino acid, any unreacted groups on the resin are blockedby treatment with excess benzoic anhydride. For each subsequent aminoacid addition to the peptide, the Fmoc group is first removed from thenascent peptide-resin complex with 15% piperidine inN-methylpyrrolidone. Coupling is performed by activation of the carboxylgroups of the N-protected amino acids with 3 mol/mol2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate/1-hydroxybenzotriazole in combination with DIPEA(diisopropylethylamine) in NMP. Activated amino acids are sequentiallyadded to the nascent peptide and mixed for 30 minutes. The extent ofcoupling is estimated by performing a ninhydrin assay (Kaiser test) on afew resin beads to detect remaining unreacted amines. Side chainprotecting groups are as follows. The guanidino function of arginine, R,is protected by 2,2,7,8-pentamethylchroman-6-sulfonyl (Pmc)), the?-amino groups of lysine (K) by t-butyloxycarbonyl (Boc), thecarboxamides of asparagine (N) and glutamine (Q) and the thiol ofcysteine by trityl (Trt) and carboxyl groups of glutamic and asparticacids as well as the hydroxyl groups of serine, threonine and tyrosineby t-butyl.

Upon completion of the synthesis, the peptide-resin is vacuum dried andcleaved and deprotected by stirring 3 hours in a solution containing 95%trifluoroacetic acid, 1% crystalline phenol, 1% ethanedithiol, 2% mlthioanisole in distilled water. The mixture is filtered through glasswool directly into ice cold diethylether. The peptide precipitate iswashed several times with additional ether and dried. Final purificationis carried out by reversed-phase HPLC on a C8 or C18 column. The purityof all synthetic peptides is ascertained by reversed phase HPLC on a C18column typically employing a water/acetonitrile gradient in the presenceof 0.1% trifluoroacetic acid. Identity is verified by MALDI-TOF massspectrometry and by amino acid composition employing a Waters Pico-TagSystem or, if necessary, by N-terminal sequence analysis. Peptidecontent is determined by quantitative amino acid analysis.

3H Thymidine assay. In particular the tritiated thymidine assays wereperformed as follows. 10,000 cells were plated in 1 ml of RPMI 1640medium containing 10% fetal bovine serum in 24-well plates. The cultureswere incubated for 24 hours at 37 C, 5% carbon dioxide. The peptide wasadded to each corresponding well at different doses and allowed toincubate for 72 hours. The cells were then exposed to tritiatedthymidine at a concentration of 1 uCi/ml (ICN, cat #2403905) andincubated at 37 C for 4 hours. The cells were washed twice with cold PBSto remove non-incorporated thymidine. The cells were treated twice with10% trichloracetic acid (Fisher, lot #94276913), 1 ml per well. Thecells were then disrupted by treatment with 10% sodium lauryl sulfate(Sigma, Cat#L-3771) at 500 ul/well. Cell from each well were transferredto a scintillation vial and counted in a Beckman Counter Model LS-133scintillation counter.

XTT method. A commercial (Roche, N.J.) kit was used. At the end of theexperiment the XTT labeling mixture was coupled to the electron reagentcoupling and was added to the cultured cells in microplate for 4 hours.The microplate was placed in an ELISA microplate reader (Moleculardevices, Menlo Park, Calif.).

Mitochondrial membrane potential. A stock solution of JC-1 dye (100μg/ml in DMSO) is prepared. Cells are preincubated with the peptide ofSEQ ID NO: 2 for up to 24 hours. JC-1 reagent is diluted just prior touse. For each assay, 100 μl of stock solution is diluted to 1 ml usingpre-warmed cell culture medium and the solution is vortexed. 40 μl ofdiluted JC-1 is added to each well and incubated at 37° C. in 5% CO₂incubator for 25-30 min. Medium is replaced with pre-warmed PBS. Samplesare detected by fluorescent signals by using flow cytometry resultscompared to buffer only treated controls. JC-1 reagent accumulates inthe intact mitochondria, giving off a bright red fluorescence. Inapoptotic cells, JC-1 reagent cannot accumulate in the mitochondria dueto the altered mitochondrial membrane potential.

Western blot performed with IgY. The materials used included:Tris-buffered Saline-Tween 20 solution (TBS-T): 1.21 g Tris-base (10mM), 8.77 g NaCl (150 mM) NaCl, in 1 L of H20, pH 7.4, containing 0.05%(v/v) Tween-20; non-fat dry milk; a GenWay Immunoblotting blockerreagent; HRP-conjugated goat anti-IgY Fc antibody (GenWay Catalog#:GAYFC-HRP) and the Immun-blot colorimetric assay kit (Bio-Rad).

An appropriate amount of cell lysates (1-10 ul of 0.5 mg/ml each lane)was separated using a 10-20% SDS-PAGE, followed by transfer to PVDFmembrane. The membrane was blocked with 5% non-fat milk in TBS-T(Tris-buffered saline containing 0.05% Tween, pH 7.4) for 1 hour at roomtemperature or longer at 4° C. (BSA is not recommended as a blockingreagent). The membrane was rinsed with TBS-T and incubated with IgYantibodies at an appropriate dilution with milk to 1% in TBS-T at roomtemperature (RT) for 1 h. Optionally the membrane can be pre-incubatedwith IgY diluted with GenWay Immunoblotting blocker at RT for 1 h priorto submerging the membrane. This step may help to reduce background,especially when E. coli-derived antigen is used. The membrane is washedwith TBS-T, 3 min each, a total of 3 times, followed by incubation withthe 2^(nd) antibody (goat-anti-IgY/Fc-HRP) at a dilution of 1:1,000 forcolorimetric assay or 1:10,000-1:100,000 for ECL (with 1% milk T BS-T)at R.T. for 1 h. The membrane is then washed with TBS-T, 3-5 min eachwith shaking, total of 3 times. Color development is performed or ECLdetection of the signal using Pierce ECL kit.

Example 2 HPLC Purified Developmental Peptides Potent AntiproliferativeEffects

HPLC, RP HPLC, etc, for example, provides for isolation of highlypurified developmental peptides that have potent antiproliferativeeffects. The activity of purified developmental peptides <3000 Da,purified by HPLC, was shown to affect several aspects of proliferation.Similar effects were noted also with <10,000 dalton developmentalpeptides. Developmental peptides <3000 Da from porcine liver induceapoptosis and activate caspases activity of preneoplastic breast cancercells. Developmental peptides also cause mitochondrial collapse with therelease Cytochrome C in MCF10neo cells. In all cases developmentalpeptides had over 63% inhibition of cell proliferation compared tocontrols.

Developmental peptides <3000 Da also inhibit 99% 3H thymidineincorporation into cellular DNA of a pre-neoplastic cell line (MCF10neocells) in a dose dependent manner. The EC50 is about 1/13^(th) Dpadilution.

Developmental peptides <3000 Da from porcine liver exert a majorinhibitory effect on resistant prostate cancer cell lines (DU145,androgen receptor negative resistant) causing apoptosis, as evidenced bythree different models of flow cytometry: a TUNEL assay, caspaseactivity as determined by cell permeability using fluorogenic D₂Rsubstrate, and mitochondrial permeability transition assessed by DiOC₆staining. With the first two methods >95% inhibition was noted comparedto controls.

FIG. 1 shows that developmental peptides are also highly effectiveagainst a surgical specimen of renal cancer cells that is highlyresistant to chemotherapy. Cells were incubated with purifieddevelopmental peptides for approximately 24-30 hrs. While developmentalpeptides were effective in inhibiting proliferation of the renal cancercells, two standardly used chemotherapies (a 5-FU regimen and avinblastine regimen) remained ineffective, even at a high concentrationsof approximately 10-100 fold.

Overall, developmental peptides exhibited an antiproliferative activityagainst an array of early stage to resistant cancer cell lines, steroidreceptor positive or negative, and actual patient tumor cells. Theeffect on cell proliferation was observed using 3H thymidine, XTT, JC-1(for mitochondrial collapse assay), P1 staining, and apoptotic pathwaysin both a time and dose dependent manner with at least >70% reduction incell survival, as documented by flow cytometry as well as bymicrophotomicrographs.

Exposure of the cells to developmental peptides caused a 99% inhibitionof proliferation, in comparison to chemotherapy, which had only amarginal effect. FIG. 2 further confirms developmental peptideseffectiveness as compared to chemotherapy. Short-term cultured renalcarcinoma cells (RCC) were incubated with developmental peptides.Analysis of cell viability using P1 staining revealed that there was aprofound loss of cell viability. It should be noted that conventionalchemotherapeutic agents, such as etoposide, paclitaxel, and methotrexatefor example, failed to induce cell death in the RCC cell lines. Whilenot wishing to be bound to theory, the anti-proliferative effect ofdevelopmental peptides is thought to be due to the developmentalpeptides induction of mitochondrial alterations in the malignant cells,as analyzed using JC-1 staining, a process which invariably leads tocell death. Preincubation of DU-145 cells, a human prostate cancer cellline, with non-lethal concentrations of developmental peptides increasedthe prostate cell sensitivity to cell death if subsequently stimulatedwith either TNF-α or paclitaxel. Thus, low concentrations ofdevelopmental peptides may enhance the efficacy of a chemotherapyregimen.

FIG. 3 illustrates that developmental peptides have a wide range ofantiproliferative activity, showing NFkβ inhibition and inhibitingangiogenic factor VEGF secretion by prostate cancer cells. Developmentalpeptides drastically reduced VEGF expression by PC-3 prostate cancercells in vitro. Developmental peptides suppress NF-kβ activity and VEGFexpression in prostate cancer cells, thereby inhibiting theirtumorigenic and metastatic properties in vitro and in vivo.Developmental peptides may diminish the angiogenic and metatstaticpotentials of prostate cells via down-regulation of NF-kβ regulatedmolecules, such as, but not limited to, VEGF, IL-6, IL-8, and MMP-9.

FIG. 4 illustrates that developmental peptides cause shrinking(implosion) and loss of cell to cell contact of various cancer celllines. A similar effect was also observed using metastatic fallopiantube cancer cells (99% inhibition dose dependent).

Example 3 Antiproliferative Effects of Synthetic Developmental Peptides

HPLC, RP HPLC, and other non limiting methods can be used foridentifying developmental peptides in different tissues. The presence,isolation and characterization of one of the anti-proliferative peptidespresent in the active fraction was documented in both the adult andfetal porcine liver (SEQ ID NO: 2). A mass spectral profile of porcineembryonal liver extract revealed a number of peptides with a 970-1230dalton mass. The 970 dalton developmental peptide was sequenced usingthe Qstar Mass Spec method and its sequence was found to be novel,confirming the findings in adult liver. The partial characterization ofpotent developmental peptides at the 3-10 kDa region using fresh livertissue is described as well. Present results show that developmentalpeptides are relevant both for adult and developing tissues and theirpresence can be documented in several species making these observationsuniversal for all mammals. Thus the size of the peptides/protein are atabout 500-10,000 Da region. The actual sequence derived shares homologywith a portion of an endogenous retrovirus (FIG. 5). However, themethods described herein allow for the identification of other proteinor peptides that have antiproliferative activities.

The role of developmental peptides appears to be of high relevance,since they are produced both in the embryo as well as in the adultliver. As an example showing the wide spread developmental peptideseffect when only one compound is involved, confirmed that actuallysingle agents may have the antiproliferative activity and thereforethere is no need for using mixtures or only the animal source.

A breast cancer cell line (MCF-7) and human mammary epithelial cells(HMEC) were incubated with the 9 AA developmental peptides of SEQ ID NO:2. 3H thymidine labeling of DNA was used to analyze theanti-proliferative effect of developmental peptides using concentrationsin the range of about 1.5-12 mg/ml developmental peptides. Anantiproliferative response was maintained on the breast cancer cell line(MCF-7 cells) using low concentrations of developmental peptides in theabsence of any harmful or negative effect on the normal HMEC cells. Theantiproliferative response was repeated and analyzed using the XTTmethod. Again the 9 AA developmental peptides, at low concentrations ofabout 1.5 mg/ml, maintained the antiproliferative effect on MCF-7 cellswith minimal effect on the HMEC cells. Finally, this same analysis wasrepeated using resistant prostate cancer cells, the PC3 cell line, whichare androgen receptor negative cells. Developmental peptides was addedto the cells using concentrations of about 6-25 mg/ml for 24 hours inthe presence of 3H thymidine to measure the effect of developmentalpeptides on cell proliferation. As seen with the breast cancer cell line(MCF-7), the 9 AA developmental peptides also had an antiproliferativeeffect (approximately 98% inhibition) on the resistant prostate cancercell line at concentrations in the range of about 12-25 mg/ml. Theseresults were further confirmed using the XTT method, by directmicroscopic observations, and by JC-1 staining to detect mitochondrialcollapse, and further confirmed by flow cytometry.

Example 4 Antibodies Against Developmental Peptides

Antibodies against developmental peptides may be produced and purified.Antibodies against a nine amino acid developmental peptide of SEQ ID NO:2 (IgY, generated in chicken) were affinity purified on a columncontaining pure developmental peptides. The antibodies generated wereused as biomarkers for determining the expression in various mammaliantissues. Developmental peptides IgY is highly specific and results inthe intense staining of the human pancreatic exocrine portion(epithelial cells) of pancreatic tissue in the absence of binding to theendocrine portion of the pancreas (Langerhans cells). The staining wasspecific in that binding of antibody to tumor cells was neutralized byaddition of the developmental peptides. Also, a control IgY wasineffective in binding to tumor cells. In contrast, the normal liverstaining was essentially negative. Developmental peptide IgY bindsnormal tissue only very weakly in the epithelial glandular layer ofvarious tissues, such as prostate, fallopian tube, and stomach, colonepithelium in human and mouse tissue arrays in a non limiting manner. Nobinding to the liver tissue was detected. Other tissues of normal humanand mouse did not bind to developmental peptide IgY. The presence ofdevelopmental peptides in epithelial cells reflect a strategiclocalization of the nine amino acid developmental peptides and relatedproteins in cells that are most liable to become malignant (that is,those cells that are normally proliferating), and thus may createhomeostasis by counteracting local growth factors. Altered proliferationmay become a cancer under the influence of mitogens, carcinogen, oraltered gene expression is prevented.

Evidence that the nine amino acid developmental peptides antibodyreflects staining of specific proteins as shown by Western blot. Humanplacental extracts, as well as the pancreas and liver, expresseddevelopmental peptides of SEQ ID NO: 2 related pro-proteins, confirmingthat the immunohistochemistry actually reflects expression ofdevelopmental peptides and related proteins. The developmental peptideIgY stained the hepatocarcinoma sample in contrast to normal liver(which was negative). This increased staining was confirmed in severalhuman tumors from almost all organs. Anti-developmental peptide IgYspecifically binds to adult human bladder cancer tissue, adult humanlung carcinoma tissue, cervical cancer, and endometrial carcinoma tissuewith almost no binding to normal endometrium. The Western blot patternin placental, pancreas and liver tissue lysates revealed expression ofprotein bands about 20-40 kDa, similar to the human placenta, pancreasand normal liver. However, the first trimester blot contained only oneband at about 15 kDa region, indicating differential expression duringpregnancy. The pattern detected in liver cancer revealed high molecularweight (40-140 Da) proteins that does not match any observed in normalliver or pancreas, while the 32 kDa size protein was attenuated incancer. Altered proteolytic processes that impair active developmentalpeptide formation may contribute to the tumor pathogenesis.

Overall, generation of the IgY antibody documented by two independentmethods demonstrate that developmental peptide related proteins andpeptides are differentially expressed in tumors compared with normaltissues. Discovery of the altered protein synthesis and degradation maybe of high relevance for understanding tumor propensity in that thegreat majority of cases of tumorigenesis are of epithelial origin.Additional observations using the tumor array revealed that in almostall instances there was an intense staining of the blood vessels, whichare known to proliferate intensely although aberrantly duringmalignancy, is further documentation that the antibody targets rapidlydividing cells. The blood vessels are the prime source of nourishmentand growth of the tumor. Indeed, lack of vascularity greatly limits theability of the tumor to grow beyond a certain size. Thus, both theepithelium and the blood vessels appear to be the major sites where theexpression of developmental peptides, including the developmentalpeptides of SEQ ID NO: 2, and related proteins are responsible forcontrol of proliferation. A detailed observation of the microphotographalso documents that the antibody binds practically only to thecytoplasmic region of the cells.

Example 5 Developmental Peptide Receptors

The developmental peptides and related molecules act through specificsites of action which may be located within the cell and are selectivelyactivated by cellular transformation. This is shown by lack of uptake ofFITC developmental peptides by normal rat embryos in culture. Incontrast, the labeled developmental peptides bound to embryonic tissuereceptors following exposure to mutagen, such as for example 5FU. Thisindicates that the receptor for developmental peptides exists inembryonic tissues however it becomes accessible to the peptide only whenthere is exposure to a mutagen/carcinogen. Thus, the production ofdevelopmental peptides occurs in embryonic tissues as well where thetarget organs are local but are only activated, in the case of exposureto an adverse environment, thereby protecting and likely eliminatingonly abnormal cells through apoptosis or non apoptotic pathways withoutaffecting normal cells. Therefore the use of developmental peptidescould identify and characterize such receptors leading to their cloningand expression. This could be achieved by using enriched membranes wherethe receptor is highly expressed (embryos following exposure to 5FU, asa non limiting example) followed by using an affinity chromatographycontaining a Biotin labeled developmental peptides column followed bymass spectometry and sequencing. In addition, characterization of thereceptor could lead to the identification of the specific intracellularmechanism and the transduction molecules that create cell death, therebyrevealing fundamental properties that the abnormal cells possess thatcreate an integrated response to the presence of the developmentalpeptides overcoming resistance to cell death that is seen withchemotherapy. These new and integrated transduction mechanismsidentified could lead to further development and design of newpharmacophores and targeted drugs.

Since one aim may be to develop cancer drugs targeted for human therapyand since the human pancreas and placenta shared similar protein patternas observed by Western blot, an affinity chromatography using thedevelopmental peptide IgY column was designed. The protein profile inthe liver cancer sample was different as compared to normal tissues,which may be due to abnormal synthesis, processing, and/or degradation.High molecular weight proteins were exclusively found in the liver tumor(approximate MW of 140, 90, 60) with attenuation of the 30 kDa band. Anumber of proteins were identified by mass spec analysis ofnon-denatured human placental tissue extracts using the developmentalpeptide IgY antibody. The major protein observed was 32 kDa, similar tothat seen by Western blot. The partial sequence and comparison withknown protein using mass spec based sequencing revealed that the majorprotein may be attached to non muscle actin (gamma actin) and to aportion of RNA polymerase molecule, both of which are known to interactwith HERV. Further analysis of the <10 kDa region revealed that themajor protein may be a portion of HERV (gag, or env portion) confirmingthe earlier observations with the developmental peptides of SEQ ID NO: 2homology to HERV pol. Thus, in the placenta the developmental peptidesappear to be attached to the cytoskeleton of the cells which are locatedin the cytoplasm of the cell. Such observations suggest a linkagebetween cell proliferation and cytoskeleton architecture, and cellmobility and implicates the developmental peptide as a likely partner inregulating cytoskeleton function which is altered in malignancy andfollowing viral infection. This data further documents that HERVs arelikely to have multiple roles beyond the immune suppression identifiedto date.

Thus, the methodology described allows for the isolation of cellularproteins and possibly other transcription factors and regulatory agentsthat are operative during development as well as in the adult and arepresent mainly in epithelial cells. The method described also allows foridentification of various proteins that are over-expressed in cancercells and tissues and others that are suppressed using, for example,Western blot, 2 D gel electrophoresis, Mass spectometry, and affinitychromatography as described in a non limiting manner.

The data generated allows further separation of the large proteins fromthe cytoskeleton elements to derive the active developmental peptidesthat are expected to have a major anticancer effect approximately 1000fold greater than the effectiveness of the developmental peptides of SEQID NO: 2 and which could be used for the prevention and treatment ofproliferative disorders as well as diagnosis of such disorders. Inaddition, identification of specific interacting factors withdevelopmental peptides can be applied for both diagnostic andtherapeutic application by using the compounds themselves or bygenerating antibodies to test for those in tissues and biological fluidsin a non-limiting manner.

In a further examples, <10 kDa adult porcine liver developmentalpeptides also have antiviral effects as shown by protection against celldeath of MRC-5 human fibroblasts that were infected withcytomegalovirus. These observations were documented by light microscopeas well as by XTT and X-Gal methods, the effect being dose dependent.This further substantiates earlier observations made in a previousapplication where a wide range of antiviral effects were noted with highmolecular weight developmental peptides and low molecular weight (<8000Da) developmental peptides with an HBL-100 cell line transformed withSV-40 viral protein. In addition, <10,000 kDa had significant dosedependent protective effects on Hep2 cells in culture that were exposedto respiratory syncitial virus (RSV). Protective effects were also foundagainst vaccinia virus (a small pox homologue). Preincubation with <10kDa developmental peptide results in an almost complete protectionagainst viral infection.

Extraction of the lipids from the <3 kDa fraction did not have an effecton developmental peptide antiproliferative activity. In addition, theporcine liver 3-10 kDa fraction was separated on an organomercurialagarose gel to determine if the developmental peptides have freesulphydrils. The developmental peptides retained their anticanceractivity indicating that developmental peptides do not have freesulphydrils.

Using specific inhibitors the mechanisms by which developmental peptidesblock MAPkinase was examined. It was found that the ras-raf pathway, IP3kinase, and src kinase and tyrosine phosphatases are involved indevelopmental peptide action. The transmembranal tyrosine kinase, andPKC do not appeared to be involved. Also based on additional experimentsdevelopmental peptide action is not exerted through TNF-α, TRAIL or CD95pathways.

Since developmental peptide binds only abnormal cells in the embryo aFITC-labeled developmental peptides method could be used for screeningof teratogens/mutagens. This method could identify potentialteratogens/mutagens and allow for their removal from the environment,thus preventing exposure of the fetus to the teratogen/mutagen,preventing an adverse pregnancy. In addition, such a method could beused to assess various compounds that are being developed as drugs andtherefore using as a rapid screening method to determine which of thesedrugs could be least likely to have a toxic effect while maintaining itspotential therapeutic profile.

Example 6 Sequence Analysis of Human Protein Database

A blast search was performed Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr (SEQ IDNo.2) versus the human protein database. Table 1 illustrates the humanprotein matches. Table 2 illustrates the amino acid distributionfrequency across aligned sequences and upstream/downstream sequences forthe matches.

TABLE 1 SEQUENCING OF THE >10 Kd MW SIZE DP PROTEINS USING LC/MASSSPECTRUM METHOD Delta Scan(s) m/z Charge Sequence Reference DatabaseXCorr Cn Sp RSp 113 1461-1468 1791.956 2

-SYELPDGQVITIGNER.F gi|998467|gb| human_0-10 3.913 0.712 1336.3AAB34251.1|? kD.fast  88 1348-1358 946.137 2 R.AVFPSIVGR.P gi|98468|gb|human_0-10 2.322 0.668 607.8 AAB34252.1|? kD.fast  95 1364 1956.206 2K.LKEQYVNKTIVFNQSSG gi|4580115|gb| human_0-10 2.147 0.532 183.4AAD24254.? kD.fast  97 1366-1380 1958.138 3 K.SDVVQtDNNKNYTKYR.

gi|857635|gb| human_0-10 1.789 0.122 172.4 AAC54509.1|? kD.fast  651252-1276 1906.186 3 K.AAYLQETGKPLDETLKK gi|6729710|pdb| human_0-101.733 0.249 314.9 1BO9|A kD.fast 111 1448-1456 1959.169 2

-.CTINANYGNM#DTEM#V gi|3065485|gb| human-0-10 1.656 0.022 359.6AAC61162.? kD.fast  87 1333-1349 1842.019 3 R.RAEPAADGVGAVSRDLgi|424402|gb| human_0-10 1.519 0.250 667.9 AAA44245.1|? kD.fast 459 31901853.155 3

-.DRLHPVHAGPIAPGQMR gi|4098015|gb| human_0-10 1.480 0.323 143.0AAD00191.? kD.fast  78 1296-1302 1905.034 3 R.GLVLATNNQDNPHPQGgi|485020|pir| human_0-10 1.449 0.208 446.5 P10548 kD.fast 578 3441-34651968.259 3 R.AFYASRQIGDMRQAHC gi|415141|gb| human_0-10 1.445 0.202 70.3AAA44354.1|? kD.fast 175 1745-1752 1980.363 3 R.VLAEAMSQVQNAAIMM

gi|3462724|gb| human_0-10 1.444 0.060 310.3 AAC33060.? kD.fast 5333341-3369 1938.174 3 R.AYCNVNRAAWNETLRR gi|15429907|gb| human_0-10 1.4380.045 27.0 AAK98388 kD.fast 100 1382 1955.178 2 K.LREQFNXTTIVFNQSSG.

gi|8489575|gb| human_0-10 1.427 0.243 85.9 AAF75719.? kD.fast 461 31931858.250 3 K.VIVQLNETVQIKCTR.P gi|2570736|gb| human_0-10 1.415 0.37892.0 AAB82244.? kD.fast 633 3538-3572 1311.389 3 R.AFNC^(±)HVEYEK.Tgi|10720143|sp| human_0-10 1.395 0.154 161.2 O91518|N kD.fast ReferenceScore Hits Entries   1 gi|6435104|gb|AAF08451.1|(AF152813) envelopeprotein 20.0 20000  603 630 :::: [Human immunodeficie   2gi|11875564|gb|AAG40705.1| 20.0 20000   89 481::::   3gi|998468|gb|AAB34252.1|48 kda histamine receptor 20.0 20000   88 90::::subunit peptide 1 (inte   4 gi|1703643|gb|AAB37684.1|laminin alpha2-chain short 18.0 11000  541:630::: arm (cysteine-rich r   5gi|3462724|gb|AAC33060.1|(AF082448) gag protein 16.0 10100  175::175::[Human immunodeficiency v   6 gi|10863925|ref|NP_066951.1|(NM_021138)DNa directed 14.0 12411 :578:578:: RNa polymerase I po   7gi|7245807|pdb|1DV0|A Chain A, Refined Nmr Solution 14.0 11100 :95:97::Structure Of The C-Ter   8 gi|4580121|gb|AAD24257.1|(AF095023) envelope12.0 10002  475::::572 glycoprotein [Human immunode

indicates data missing or illegible when filed

TABLE 2 AMINO ACID RESIDUE DISTRIBUTION FREQUENCY ACROSS ALIGNEDSEQUENCES AND UPSTREAM/DOWNSTREAM SEQUENCES Peptide sequence 1 V L G K R2 Residue Pos1 Pos2 Pos3 Pos4 Pos5 Pos6 Pos7 Pos8 Pos9 Pos10 Pos11 3 A 80 1 2 3 0 5 0 9 0 0 4 C 2 1 0 0 0 2 0 2 1 0 0 5 D 4 5 5 5 3 0 1 0 1 0 06 E 1 9 4 3 3 3 3 1 1 0 0 7 F 2 3 4 2 2 1 4 0 1 0 0 8 G 1 5 3 3 9 0 1 025 0 1 9 H 3 3 0 2 4 2 2 0 0 0 0 10 I 0 1 2 7 4 3 6 2 0 0 1 11 K 4 3 2 41 3 2 0 0 43 4 12 L 7 1 4 2 7 0 2 38 2 0 0 13 M 1 2 1 1 1 2 0 2 0 0 0 14N 0 1 2 3 2 3 1 0 1 0 0 15 P 2 1 4 1 3 7 2 1 0 1 0 16 Q 1 5 3 3 3 5 1 20 0 1 17 R 1 1 3 1 1 3 0 0 1 4 41 18 S 6 3 4 3 0 3 4 1 6 1 0 19 T 3 2 12 0 3 2 0 0 0 0 20 V 1 2 4 4 3 7 12 0 0 0 1 21 W 2 0 0 0 0 1 1 0 1 0 022 Y 0 1 2 1 0 1 0 0 0 0 0 23 Peptide sequence 1 I K G T 2 Pos12 Pos13Pos14 Pos15 Pos16 Pos17 Pos18 Pos19 3 0 0 3 2 2 4 2 4 4 0 0 1 0 1 1 1 05 0 2 2 1 1 3 5 2 6 1 1 0 3 1 8 3 1 7 1 0 0 3 3 1 0 0 8 0 2 11 1 9 6 4 79 0 0 0 0 2 1 3 1 10 36 2 1 8 5 0 3 6 11 0 32 1 4 5 8 3 3 12 5 0 1 3 2 16 3 13 0 1 1 2 0 1 0 1 14 0 3 2 1 1 1 0 2 15 0 0 2 0 1 3 4 0 16 0 0 2 11 2 2 3 17 0 0 7 8 0 3 1 1 18 0 5 2 3 5 3 7 6 19 1 1 1 7 4 2 1 4 20 4 011 1 3 0 1 5 21 0 0 0 0 2 0 1 0 22 1 0 1 1 1 1 2 0 23 Table 2 is theamino acid distribution frequency across aligned sequences andupstream/downstream sequences for the human protein database matches.

Example 7 Binding of Developmental Peptides to Abnormal Embryo

Rat embryo cultures were exposed to synthetic developmental peptides ofabout 9 AA (about 500 μg/ml) for 48 hours (FIG. 6). The viscera yolk sacand the embryo both developed normally. Thus, the developmental peptidesof the present invention are not teratogenic.

Whole rat embryos (within the amniotic sac) were exposed toFITC-developmental peptides for 48 hours (FIG. 7). There was no uptakeof peptides by the tissue. In contrast, when the rat embryo's were grownin normal serum and exposed to +/−5-FU (a potentteratogen/chemotherapeutic agent) for 3 hours (FIG. 8), followed byexposure to FITC-developmental peptides for 48 hours. Fluorescentstereomicroscopy revealed that the fluorescent-developmental peptidesaccumulated in the tissues. This result reveals that developmentalpeptides bind only to specific receptors that are present or have beenactivated on damaged embryos. In this example the damage was due toexposure to the 5-FU, a potent teratogen/chemotherapeutic agent. Undernormal conditions the receptors remain non-active.

Example 8 Binding of Developmental Peptides to Proliferating Cells ofFirst Trimester Placenta

The 9 AA developmental peptide IgY antibody identifies proliferatingcells. First trimester placenta was cultured on Matrigel and stainedwith anti-developmental peptide IgY. Anti-developmental peptideantibodies bind specifically to the proliferating cells in the firsttrimester placenta, this binding is accentuated after 96 hours ofculture.

Examination of the first trimester placenta, after exposure toanti-developmental peptides IgY, revealed that only the proliferatingcells of the extravillous trophoblast (EVT) were bound byanti-developmental peptides antibodies. The EVT cells also have a highinvasive potential, however they stop proliferating once adequatecontact with the maternal endometrium is well established. Developmentalpeptides may serve to limit such invasivity in vivo. The first trimesterplacental extracts were stained with anti-HSP27 and anti-Ki27,antibodies that detect the corresponding HSP27 and Ki27, twoproliferation markers. The anti-HSP27 and anti-Ki27 antibodies boundspecifically to the proliferating cells, confirming the specificity ofthe anti-developmental peptide antibody which also bound to theproliferating cells only.

First trimester explants were exposed to either anti-developmentalpeptides and anti-HSP27 antibodies or to anti-developmental peptides andanti-Ki27 antibodies. The HSP27 and Ki27 proliferation markers wereobserved to co-localize with developmental peptides in placentalexplants. This confirms the role of developmental peptides as a negativeregulator of placental proliferation, possibly by directing cells towarddifferentiation instead of proliferation and invasivity, neoplastic-likeproperties.

Western blot analysis of normal adult and placental tissue were probedwith affinity purified developmental peptide IgY (1:500) followed bygoat ant-IgY HRP antibody 1:1000. The results revealed that termplacenta, pancreas and liver patterns were similar. Normal and cancerousliver extracts were also analyzed by Western blot (using the sameantibodies) The results revealed that the 9 AA developmental peptideantibodies identified an altered pattern of developmental peptideexpression in the cancerous tissue sample.

What has been described and illustrated herein are embodiments of theinvention along with some of their variations. The terms, descriptionsand figures used herein are set forth by way of illustration only andare not meant as limitations. Those skilled in the art will recognizethat many variations are possible within the spirit and scope of theinvention, which is intended to be defined by the following claims andtheir equivalents in which all terms are meant in their broadestreasonable sense unless otherwise indicated.

1. A method of treating a proliferative disorder in a subject comprising administering to said subject a peptide selected from the group consisting of N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 1), N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 2), N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 3), N-Ile-Asp-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 4), N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 5), N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 6), N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 7), and N-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID NO: 8).
 2. The method of claim 1, wherein said proliferative disorder is cancer.
 3. The method of claim 1, wherein said proliferative disorder is a viral infection.
 4. The method of claim 1, wherein an effective amount of said peptide is administered to said subject.
 5. The method of claim 4, wherein an effective amount of said peptide is 1 mg to 2000 mg.
 6. The method of claim 4, wherein an effective amount of said peptide is 1 mg to 1000 mg.
 7. The method of claim 4, wherein an effective amount of said peptide is 50 mg to 600 mg.
 8. The method of claim 1 further comprising administering a therapeutic agent selected from the group consisting of chemotherapeutic agents, immune modulators, and angiogenic inhibitors.
 9. The method of claim 1, wherein said peptide is administered in a composition comprising a pharmaceutical carrier.
 10. The method of claim 1, wherein said peptide is SEQ ID NO: 1 (N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 11. The method of claim 1, wherein said peptide is SEQ ID NO: 2 (N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 12. The method of claim 1, wherein said peptide is SEQ ID NO: 3 (N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 13. The method of claim 1, wherein said peptide is SEQ ID NO: 4 (N-Ile-Asp-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 14. The method of claim 1, wherein said peptide is SEQ ID NO: 5 (N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 15. The method of claim 1, wherein said peptide is SEQ ID NO: 6 (N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 16. The method of claim 1, wherein said peptide is SEQ ID NO: 7 (N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH).
 17. The method of claim 1, wherein said peptide is SEQ ID NO: 8 (N-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH). 